The need to conjugate biomolecules with labels is found in all areas of bioscience research, diagnostics and medicine. Labels that are attached to biomolecules include proteins (e.g. enzymes, fluorescent proteins, streptavidin), oligonucleotides and small-molecule ligands (SMLs; singular SML) usually with molecular weights of less than 1000 (e.g. biotin, fluorescent dyes, metal ion chelators, photoreactive groups, iodinatable molecules, photosensitisers, quenchers, short peptides and drugs). SMLs that are employed in conjugation reactions usually, but not always, have a reactive (often amine-reactive [AR]) group that facilitates attachment of the SML to the biomolecule. For example, fluorescein is normally introduced using an isothiocyanate derivative (FITC; fluorescein isothiocyanate) or an N-hydroxysuccinimide (NHS) derivative.
The majority of activated SMLs used in conjugation reactions are NHS esters, which have a number of attractive features, such as facile coupling to amines at physiological pH. This generates an amide link that is both strong and irreversible. Disadvantageously, NHS esters are prone to decomposition on storage, especially if moisture enters the product, and they are very susceptible to hydrolysis in aqueous solutions. As SMLs are monovalent with respect to the reactive moiety, the loss of any reactive groups reduces the percentage of SML that can participate in conjugation reactions. Because of possible decomposition on storage and because of competing hydrolysis reactions, the SML is generally used in a significant molar excess. As many biomolecules contain multiple amine functions, trial experiments are usually carried out on a small scale with varying molar ratios and/or varying reaction times to optimise the conditions and to avoid over-labelling. Finally, with the need to use a significant excess of SML, it is inevitable that large amounts of unconjugated SML and/or hydrolysis products will contaminate the final product, and that purification of the conjugate will be required.
An alternative chemistry that avoids some of the problems encountered with NHS esters involves thiol-mediated coupling of molecules. Thiol-reactive (TR) SMLs are relatively stable but because of their more limited commercial availability and because many biomolecules lack of indigenous free thiols TR-SMLs are less frequently used in conjugation reactions. While methods to introduce thiols into biomolecules are known, the operation of adding thiol groups usually makes the process of conjugation technically more complicated. Functional groups found in commercially available SMLs that react with thiol groups include maleimide, iodoacetyl, bromoacetyl, aziridine, epoxide, acryloyl, and thiol-disulfide exchange reagents (e.g. pyridyl disulfides).
The present invention relates to, inter alia, methods for making conjugates that circumvent at least some of the problems associated with reactive SMLs, particularly NHS esters, and with other SMLs that are monovalent with respect to their reactive functional groups. These methods are described more fully below.